-
Cell Reports Jun 2024The protein phosphatase 2A (PP2A) regulatory subunit PPP2R2A is involved in the regulation of immune response. We report that lupus-prone mice with T cells deficient in...
The protein phosphatase 2A (PP2A) regulatory subunit PPP2R2A is involved in the regulation of immune response. We report that lupus-prone mice with T cells deficient in PPP2R2A display less autoimmunity and nephritis. PPP2R2A deficiency promotes NAD biosynthesis through the nicotinamide riboside (NR)-directed salvage pathway in T cells. NR inhibits murine Th17 and promotes Treg cell differentiation, in vitro, by PΑRylating histone H1.2 and causing its reduced occupancy in the Foxp3 loci and increased occupancy in the Il17a loci, leading to increased Foxp3 and decreased Il17a transcription. NR treatment suppresses disease in MRL.lpr mice and restores NAD-dependent poly [ADP-ribose] polymerase 1 (PARP1) activity in CD4 T cells from patients with systemic lupus erythematosus (SLE), while reducing interferon (IFN)-γ and interleukin (IL)-17 production. We conclude that PPP2R2A controls the level of NAD through the NR-directed salvage pathway and promotes systemic autoimmunity. Translationally, NR suppresses lupus nephritis in mice and limits the production of proinflammatory cytokines by SLE T cells.
PubMed: 38889006
DOI: 10.1016/j.celrep.2024.114379 -
Investigative Ophthalmology & Visual... Jun 2024Ubiquitination serves as a fundamental post-translational modification in numerous cellular events. Yet, its role in regulating corneal epithelial wound healing (CEWH)...
PURPOSE
Ubiquitination serves as a fundamental post-translational modification in numerous cellular events. Yet, its role in regulating corneal epithelial wound healing (CEWH) remains elusive. This study endeavored to determine the function and mechanism of ubiquitination in CEWH.
METHODS
Western blot and immunoprecipitation were used to discern ubiquitination alterations during CEWH in mice. Interventions, including neuronally expressed developmentally downregulated 4 (Nedd4) siRNA and proteasome/lysosome inhibitor, assessed their impact on CEWH. In vitro analyses, such as the scratch wound assay, MTS assay, and EdU staining, were conducted to gauge cell migration and proliferation in human corneal epithelial cells (HCECs). Moreover, transfection of miR-30/200 coupled with a luciferase activity assay ascertained their regulatory mechanism on Nedd4.
RESULTS
Global ubiquitination levels were markedly increased during the mouse CEWH. Importantly, the application of either proteasomal or lysosomal inhibitors notably impeded the healing process both in vivo and in vitro. Furthermore, Nedd4 was identified as an essential E3 ligase for CEWH. Nedd4 expression was significantly upregulated during CEWH. In vivo studies revealed that downregulation of Nedd4 substantially delayed CEWH, whereas further investigations underscored its role in regulating cell proliferation and migration, through the Stat3 pathway by targeting phosphatase and tensin homolog (PTEN). Notably, our findings pinpointed miR-30/200 family members as direct regulators of Nedd4.
CONCLUSIONS
Ubiquitination holds pivotal significance in orchestrating CEWH. The critical E3 ligase Nedd4, under the regulatory purview of miR-30 and miR-200, facilitates CEWH through PTEN-mediated Stat3 signaling. This revelation sheds light on a prospective therapeutic target within the realm of CEWH.
Topics: Nedd4 Ubiquitin Protein Ligases; Animals; Ubiquitination; Mice; Cell Movement; Cell Proliferation; Wound Healing; PTEN Phosphohydrolase; Epithelium, Corneal; Ubiquitin-Protein Ligases; Humans; Mice, Inbred C57BL; Endosomal Sorting Complexes Required for Transport; Blotting, Western; STAT3 Transcription Factor; Cells, Cultured; Disease Models, Animal; MicroRNAs; Immunoprecipitation; Male; Gene Expression Regulation
PubMed: 38888282
DOI: 10.1167/iovs.65.6.29 -
Journal of Extracellular Vesicles Jun 2024Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent...
Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.
Topics: Microglia; Extracellular Vesicles; Nitric Oxide; Animals; Lipopolysaccharides; Signal Transduction; Mice; Proteomics; Protein Processing, Post-Translational; Cysteine; Nitric Oxide Synthase Type II
PubMed: 38887871
DOI: 10.1002/jev2.12455 -
PeerJ 2024Despite extensive research highlighting the pivotal role of MYB transcription factors in regulating anthocyanin biosynthesis, the interactive regulatory network...
Despite extensive research highlighting the pivotal role of MYB transcription factors in regulating anthocyanin biosynthesis, the interactive regulatory network involving these MYB factors in pear fruits remains inadequately characterized. In this study, the anthocyanin-regulatory gene was successfully cloned from 'Yuluxiang' pear () fruits, and its influence on anthocyanin accumulation was confirmed through transient expression assays. Specifically, the co-transformation of with its partner in pears served to validate the functional role of . Subsequently, PbrMYB114 was employed as bait in a yeast two-hybrid screening assay, using a 'Yuluxiang' pear protein library, which led to the identification of 25 interacting proteins. Further validation of the interactions between PbrMYB114 and PbrMT2/PbrMT3 was conducted. Investigations into the role of and in 'Duli' seedlings () revealed their potential to enhance anthocyanin accumulation. The outcomes of these studies provide novel insights into the protein network that regulates pear anthocyanin biosynthesis, particularly the functional interactions among PbrMYB114 and associated proteins.
Topics: Pyrus; Anthocyanins; Transcription Factors; Plant Proteins; Gene Expression Regulation, Plant; Two-Hybrid System Techniques; Fruit
PubMed: 38887620
DOI: 10.7717/peerj.17540 -
Frontiers in Plant Science 2024Bud dormancy is crucial for woody perennial plants to resist low-temperature stress in winter. However, the molecular regulatory mechanisms underlying bud dormancy...
Bud dormancy is crucial for woody perennial plants to resist low-temperature stress in winter. However, the molecular regulatory mechanisms underlying bud dormancy release are largely unclear. Here, a tree peony () transcript (), encoding a RING-H2 finger protein, was selected from previously generated RNA sequencing data of chilling-treated buds. The objective of this study is to investigate the role of PsATL33 in the regulation of cold-induced bud dormancy release. Subcellular localization assay revealed that PsATL33 was localized to the nucleus and plasma membrane. Reverse transcription-quantitative PCR analysis showed that was dramatically upregulated during cold-triggered bud dormancy release. Exogenous treatments with gibberellin (GA) increased, but abscisic acid (ABA) inhibited the transcription of . Ectopic transformation assay indicated that overexpression of in petunia promoted seed germination, plant growth, and axillary bud break. Silencing of in tree peony through virus-induced gene silencing assay delayed bud dormancy release. tobacco rattle virus (TRV)--infected buds exhibited reduced expression levels of dormancy break-related genes () and (). Silencing of decreased the accumulation of bioactive GAs, GA and GA, rather than ABA. Transcript levels of several genes involved in GA biosynthesis and signaling, including (), (), , (), and (), were changed by silencing. Taken together, our data suggest that PsATL33 functions as a positive regulator of cold-induced bud dormancy release by modulating GA production.
PubMed: 38887463
DOI: 10.3389/fpls.2024.1395530 -
Veterinary Research Jun 2024Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune...
Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune response. The cGAS-STING signalling pathway plays an important role in antiviral innate immunity. However, it remains unclear whether PDCoV achieves immune evasion by regulating the cGAS-STING pathway. Here, we demonstrated that the nonstructural protein 2 (nsp2) encoded by PDCoV inhibits cGAS-STING-mediated type I and III interferon (IFN) responses via the regulation of porcine STING (pSTING) stability. Mechanistically, ectopically expressed PDCoV nsp2 was found to interact with the N-terminal region of pSTING. Consequently, pSTING was degraded through K48-linked ubiquitination and the proteasomal pathway, leading to the disruption of cGAS-STING signalling. Furthermore, K150 and K236 of pSTING were identified as crucial residues for nsp2-mediated ubiquitination and degradation. In summary, our findings provide a basis for elucidating the immune evasion mechanism of PDCoV and will contribute to the development of targets for anti-coronavirus drugs.
Topics: Animals; Swine; Viral Nonstructural Proteins; Deltacoronavirus; Swine Diseases; Membrane Proteins; Coronavirus Infections; Interferon Type I; Immunity, Innate; HEK293 Cells; Immune Evasion; Ubiquitination
PubMed: 38886840
DOI: 10.1186/s13567-024-01330-w -
Biology Direct Jun 2024Long noncoding RNAs (lncRNAs) are implicated in the initiation and progression of diffuse large B-cell lymphoma (DLBCL). Small nucleolar RNA host gene 20 (SNHG20) has...
BACKGROUND
Long noncoding RNAs (lncRNAs) are implicated in the initiation and progression of diffuse large B-cell lymphoma (DLBCL). Small nucleolar RNA host gene 20 (SNHG20) has been recognized as a critical lncRNA in multiple human cancers. However, the role of SNHG20 and its underlying mechanism in DLBCL are still unclear.
METHODS
The expression levels of SNHG20, c-MYC, β-catenin, and ubiquitin-specific peptidase 14 (USP14) were measured by reverse transcription-quantitative polymerase chain reaction (RT‒qPCR) and immunoblotting. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) incorporation, and flow cytometry assays were used to assess the proliferation and apoptosis of DLBCL cells. The transcriptional regulation of SNHG20 by c-MYC was confirmed by a luciferase reporter assay and RNA immunoprecipitation. The interaction between USP14 and β-catenin was demonstrated using coimmunoprecipitation. A subcutaneous xenograft model was constructed to determine the role of SNHG20 in vivo.
RESULTS
In the present study, we found that SNHG20 expression was upregulated in DLBCL cell lines and tissues compared to their normal counterparts. SNHG20 knockdown prominently reduced the proliferation and induced the apoptosis of U2932 and OCI-LY3 cells. However, SNHG20 overexpression increased the proliferation and apoptosis resistance of DLBCL cells. Mechanistically, the expression of SNHG20 was positively regulated by c-MYC in DLBCL cells. C-MYC directly bound to the promoter of SNHG20 to activate its transcription. SNHG20 was expressed mainly in the cytosol in DLBCL cells. SNHG20 silencing did not impact USP14 expression but markedly decreased the level of β-catenin, the substrate of USP14, in DLBCL cells. USP14 overexpression increased the β-catenin level, and this increase was attenuated by SNHG20 knockdown. Treatment with the proteasome inhibitor MG132 abolished SNHG20 knockdown-induced β-catenin downregulation. Moreover, SNHG20 silencing reduced the half-life but increased the ubiquitination of β-catenin in DLBCL cells. SNHG20 knockdown weakened the interaction between both endogenous and exogenous USP14 and β-catenin. In turn, SNHG20 overexpression increased the c-MYC level, and this increase was attenuated by β-catenin knockdown. Importantly, β-catenin knockdown attenuated the SNHG20-mediated increase in DLBCL cell proliferation in vitro and tumour growth in vivo.
CONCLUSIONS
Taken together, our results suggested that c-MYC-activated SNHG20 accelerated the proliferation and increased the apoptosis resistance of DLBCL cells via USP14-mediated deubiquitination of β-catenin. The c-MYC/SNHG20 positive feedback loop may be a new target for anti-DLBCL treatment.
Topics: RNA, Long Noncoding; Humans; beta Catenin; Cell Proliferation; Lymphoma, Large B-Cell, Diffuse; Proto-Oncogene Proteins c-myc; Cell Line, Tumor; Animals; Mice; Ubiquitination; Ubiquitin Thiolesterase; Gene Expression Regulation, Neoplastic; Apoptosis; Mice, Nude
PubMed: 38886753
DOI: 10.1186/s13062-024-00488-9 -
Scientific Reports Jun 2024Colon adenocarcinoma (COAD) is the second leading cause of cancer death, and there is still a lack of diagnostic biomarkers and therapeutic targets. In this study,...
Colon adenocarcinoma (COAD) is the second leading cause of cancer death, and there is still a lack of diagnostic biomarkers and therapeutic targets. In this study, bioinformatics analysis of the TCGA database was used to obtain RUNX1, a gene with prognostic value in COAD. RUNX1 plays an important role in many malignancies, and its molecular regulatory mechanisms in COAD remain to be fully understood. To explore the physiological role of RUNX1, we performed functional analyses, such as CCK-8, colony formation and migration assays. In addition, we investigated the underlying mechanisms using transcriptome sequencing and chromatin immunoprecipitation assays. RUNX1 is highly expressed in COAD patients and significantly correlates with survival. Silencing of RUNX1 significantly slowed down the proliferation and migratory capacity of COAD cells. Furthermore, we demonstrate that CDC20 and MCM2 may be target genes of RUNX1, and that RUNX1 may be physically linked to the deubiquitinating enzyme USP31, which mediates the upregulation of RUNX1 protein to promote transcriptional function. Our results may provide new insights into the mechanism of action of RUNX1 in COAD and reveal potential therapeutic targets for this disease.
Topics: Humans; Core Binding Factor Alpha 2 Subunit; Cdc20 Proteins; Ubiquitination; Gene Expression Regulation, Neoplastic; Minichromosome Maintenance Complex Component 2; Cell Line, Tumor; Colonic Neoplasms; Cell Proliferation; Ubiquitin-Specific Proteases; Disease Progression; Cell Movement
PubMed: 38886545
DOI: 10.1038/s41598-024-64726-w -
Cell Death & Disease Jun 2024TGF-β1 plays a pivotal role in the metastatic cascade of malignant neoplasms. N6-methyladenosine (mA) stands as one of the most abundant modifications on the mRNA...
TGF-β1 plays a pivotal role in the metastatic cascade of malignant neoplasms. N6-methyladenosine (mA) stands as one of the most abundant modifications on the mRNA transcriptome. However, in the metastasis of gallbladder carcinoma (GBC), the effect of TGF-β1 with mRNA mA modification, especially the effect of mRNA translation efficiency associated with mA modification, remains poorly elucidated. Here we demonstrated a negative correlation between FOXA1 and TGF-β1 expression in GBC. Overexpression of FOXA1 inhibited TGF-β1-induced migration and epithelial-mesenchymal transition (EMT) in GBC cells. Mechanistically, we confirmed that TGF-β1 suppressed the translation efficiency of FOXA1 mRNA through polysome profiling analysis. Importantly, both in vivo and in vitro experiments showed that TGF-β1 promoted mA modification on the coding sequence (CDS) region of FOXA1 mRNA, which was responsible for the inhibition of FOXA1 mRNA translation by TGF-β1. We demonstrated through MeRIP and RIP assays, dual-luciferase reporter assays and site-directed mutagenesis that ALKBH5 promoted FOXA1 protein expression by inhibiting mA modification on the CDS region of FOXA1 mRNA. Moreover, TGF-β1 inhibited the binding capacity of ALKBH5 to the FOXA1 CDS region. Lastly, our study confirmed that overexpression of FOXA1 suppressed lung metastasis and EMT in a nude mice lung metastasis model. In summary, our research findings underscore the role of TGF-β1 in regulating TGF-β1/FOXA1-induced GBC EMT and metastasis by inhibiting FOXA1 translation efficiency through mA modification.
Topics: Hepatocyte Nuclear Factor 3-alpha; Humans; Transforming Growth Factor beta1; Gallbladder Neoplasms; Animals; Epithelial-Mesenchymal Transition; Cell Line, Tumor; Adenosine; Mice, Nude; Mice; Protein Biosynthesis; Neoplasm Metastasis; Gene Expression Regulation, Neoplastic; Cell Movement; RNA, Messenger; Mice, Inbred BALB C; Male
PubMed: 38886389
DOI: 10.1038/s41419-024-06800-9 -
Life Science Alliance Sep 2024Protein translation initiation is a conserved process involving many proteins acting in concert. The 13 subunit eukaryotic initiation factor 3 (eIF3) complex is...
Protein translation initiation is a conserved process involving many proteins acting in concert. The 13 subunit eukaryotic initiation factor 3 (eIF3) complex is essential for assembly of the pre-initiation complex that scans mRNA and positions ribosome at the initiation codon. We previously reported that a gain-of-function (gf) mutation affecting the G subunit of the eIF3 complex, , selectively modulates protein translation in the ventral cord cholinergic motor neurons. Here, through unbiased genetic suppressor screening, we identified that the gene mediates ()-dependent protein translation in motor neurons. LIN-66 is composed largely of low-complexity amino acid sequences with unknown functional domains. We combined bioinformatics analysis with in vivo functional dissection and identified a cold-shock domain in LIN-66 critical for its function. In cholinergic motor neurons, LIN-66 shows a close association with EIF-3.G in the cytoplasm. The low-complexity amino acid sequences of LIN-66 modulate its subcellular pattern. As cold-shock domains function broadly in RNA regulation, we propose that LIN-66 mediates stimulus-dependent protein translation by facilitating the interaction of mRNAs with EIF-3.G.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Eukaryotic Initiation Factor-3; Protein Biosynthesis; Motor Neurons; Mutation; RNA, Messenger; Amino Acid Sequence; Cold-Shock Response; Protein Domains
PubMed: 38886018
DOI: 10.26508/lsa.202402673